The present invention relates to the field of the application of recombinant DNA technology to the genetic transformation, or genetic engineering, of higher plants. More specifically, the invention relates to a strategy for effecting somatic changes in higher plants through the use of negative RNA strands, so as to control gene expression in plants or to achieve other useful somatic effects, such as disease resistance.
It has now become possible to construct fragments, of genetic material, i.e. DNA, in vitro, and to transform those fragments into plasmids contained in bacteria. It has also become possible to use cloned fragments, or to clone entire genes, in bacterial plasmids known as vectors, which can carry those fragments, or genes into other cells. Suitable vectors have been developed which can be used to genetically transform individual plant cells from which full intact, plants can be regenerated. It is has been documented that foreign genes can be stably inserted into the genome of plant cells, and that whole, intact, somatically normal and reproductively competent plants can be reconstructed therefrom. K. A. Barton, et al., Regeneration of Intact Tobacco Plants Containing Full-Length Copies of Genetically Engineered T-DNA, and Transmission of T-DNA to R1 Progeny, 32 Cell 1033 (April 1983). Investigators have reported that they have been able to introduce complete foreign genes into plant cells, and obtain gene expression in those plant cells, with the understanding and expectation that the cells are capable of being regenerated into whole, intact, plants. European patent application S.N. 84302582.2, filed Apr. 16, 1984 (Kemp); PCT application number W084/02920 and W084/02913, both filed Jan. 16, 1984 (Fraley). In general, inserted genes will function in plants only when constructed as chimeric insertions with proper plant gene control regions appropriate for plant systems.
Most of the current strategies used for creating genetically engineered plants involve modification on a cellular level of plant cells through the use of the natural plant transforming agent Agrobacterium tumefaciens, which has the natural ability to infect dicotyledonous plants and to transfer a certain portion of the DNA (referred to as T-DNA) of the A. tumefaciens into the plant cell. Other techniques have been proposed, not involving A. tumefaciens, for transforming individual plant cells, particularly protoplasts, of both dicots and monocots. The principle obstacle to successful genetic engineering of a wide number of plant species at present is the difficulty in regenerating many plant species from callus culture or protoplasts. For those species for which regeneration techniques are currently available, genetic transformation of cells in culture can result in full foreign gene expression in intact otherwise normal plants. For plant species for which regeneration techniques are not presently refined, once those techniques are developed, regular genetic transformation of intact plants of those plant species will become a common practice.
Once it is possible to genetically engineer a plant species, the question then becomes what logical genetic transformations can be achieved in the plant in order to make the plant more suitable for the agricultural or horticultural uses for which it is normally intended. One common strategy for the utilization of genetic engineering in plants is to introduce exogenous protein genes into plants to cause expression in the plant of a protein which may be useful for one or more purposes, such as disease resistant, insect resistance, enzymatic activity, utility as a food ingredient, etc.
The invention described here provides an alternative strategy for the use of genetic engineering techology in plants to achieve useful somatic changes to plants, not involving the expression of any exogenous proteins, but instead controlling the expression of an endogenous protein or the operation of a protein gene or other DNA or RNA factor naturally introduced into the plant cells through outside agents, such as agents of disease or infection.
It has been previously recognized that artificially constructed negative strand RNAs will hybridize with complementary RNAs in vivo. This phenomenon has been utilized to investigate the regulatory mechanisms of gene expression in E. coli. Mizuno et al, Regulation of Gene Expression by a Small RNA Transcript (mic RNA) in Escherichia coli K-12, 10 Proc. Japan Acad. 59, Ser. B, pp. 335-338 (1983), Mizuno et al, A Unique Mechanism Regulating Gene Expression: Translational Inhibition by a Complementary RNA Transcript (mic RNA), 81 Proc. Natl. Acad. Sci. USA, pp. 1966-1970 (1984).
The present invention relates to a method for performing useful genetic transformations of plants to achieve useful somatic changes in the plants themselves, not specifically involving the expression of exogenous proteins. The method involves the introduction into the plant genome of DNA sequences constructed for the transcription of negative strand RNA which is substantially complementary to target endogenous or naturally introduced RNA strands, whose function it is desired to inhibit so as to prevent either the expression of an endogenous protein gene or the operation of a naturally introduced RNA or DNA, such as occurs through certain types of parasitic or disease infection.
It is an object of the present invention to provide a strategy for genetically engineering plants to create plants having useful somatic characteristics without necessarily causing the expression of exogenous proteins.
It is another object of the present invention to provide a method for controlling endogenous gene expression in plants in general.
Other objects and advantages of the present invention will become apparent from the following specification.